Development of recombinant proteins with an aim to increase their therapeutic activity by the regulation of intracellular sorting

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Biologically active proteins usually exhibit the short plasma half-life, repeated and high doses being necessary to obtain their pharmacological activity in vivo. Since their clearance mechanism is the receptor-mediated endocytosis and subsequent lysosomal degradation in target organs, the aim of this study is to regulate their intracellular sorting in order to avoid such degradation pathway. Hepatocyte growth factor (HGF) was fused with endoplasmic reticulum retention signal and nuclear localization signal by gene recombinant techniques. HGF genes obtained in this way were inserted into PUC-SRaipha vector and transfected into COS-7 cells. HGF produced in the medium was dialyzed and purified by heparin-affinity chromatography and reverse phase HPLC. HGF was iodinated by the chloramine-T method, its molecular weight being checked by SDS-PAGE. The biological activity of each HGF derivative was checked by assessing the stimulatory effect on DNA synthesis in primary cultured rat hepatocytes. After incubating with cell lines which highly express HGF receptor, the trichloroacetic acid-soluble radioactivity in the medium gradually increased after a lag-time. However, the increase in TCA-soluble radioactivity during the incubation period with radiolabeled HGF mutant was much lower, indicating that its intracellular degradation is much slower than the wild-type HGF. Thus, the regulation of the intracellular fate of the endocytosed ligand can be, at least partially, regulated by the insertion of a certain type of signal sequences. However, the stability of such a mutant in the medium was not so much increased probably because of the low efficiency in the recycle of the ligand. Therrefore, further improvement has to be considered to change the sorting after avoiding lysosomal degradation. The dissociation kinetics in the endosomal compartment may be the other target to improve the efficiency of ligand recycle.